Enumeration of cell number and determining the spatial distribution of cells has wide applicability to biological research. It is the basis of assays for the study of compounds that promote or inhibit cell proliferation, cell adhesion, and cell migration at cell invasion into extracellular matrices. All of these in vitro assays are used in studies of fundamental biology as well as in assessing the potential therapeutic benefits of investigational compounds. Direct optical counting of cells for cell enumeration is in many respects considered the gold standard. See Sheppard, B. C., Rutten. M. J., Meichsner, C. L., Bacon, K. D., Leonetti, P. O., Land, J., Crass, R. C., Trunkey, D. D., Deveney, K. E., Deveney, C. W., Cancer, 85, 1454-64 (1999); Dunk, C., Ahmed, A., Am. J. Pathol. 158, 265-273 (2001). These methods are labor intensive, however, which has motivated the development of a wide array of indirect methods (e.g., MTT, Calcein AM which act as intracellular enzyme cleavage substrates) for use in determining cell number in assays of cell proliferation and cell migration. With few exceptions, methods integrating enumeration of cell number with spatial location available for the study of cell migration are very laborious, can consume large amounts of cells and reagents, and are generally not amenable to development of the high throughput systems needed to accelerate drug discovery and development of lead compounds for clinical applications. This is especially true for assays capable of separating out an increase in random cell movement (chemokinesis) from directed cell movement in response to a stimulus gradient (chemotaxis). Liquid crystal reporting systems have recently been disclosed for various purposes and could potentially meet all or many of these largely unmet needs. The missing element, however, in making this a truly robust technology for broad application is liquid crystals that exhibit reduced, little, or no toxicity to cells and a liquid crystalline cell culture media that supports normal cell function. Therefore, there is a need for liquid crystal compositions that exhibit reduced, little, or no toxicity to cells to which they are exposed as well as for new cell culturing media that includes liquid crystals that have reduced, little, or no toxicity to the cells being cultured or investigated.
Recently, liquid crystals have begun to be employed as novel and useful tools for application in devices for use in the physical and life sciences. Gupta, V. K., Skaife, J. J., Dubrovsky, T. B., Abbott, N. L., Science, 279, 2077 (1998); Luk, Y.-Y., Tingey, M. L., Hall, D. J., Israel, B. A., Murphy, C. J., Bertics, P. J., and Abbott, N. L., Langmuir, 19, 1671 (2003); Kim, S. R., Abbott, N. L., Langmuir, 18, 5269 (2002); Kim, S. R., Abbott, N. L., Adv. Mater., 13, 1445 (2001); Shah, R. R., Abbott, N. L., Science, 293, 1296 (2001). For example, nematic crystals have been reportedly used to amplify protein binding events on receptor decorated self-assembled monolayers (SAMs) supported on gold films that possess nanometer-scale topography. By using buffed films of biotinylated bovine serum albumin (BSA) covalently immobilized on glass substrates, liquid crystals have been exploited to detect the binding of antibody to surface bound biotins. Finally, by using surfaces that present metal ions that bind mesogens, reversible detection of parts-per-billion (by volume) levels of chemical agents such as organophosphonates has been established. See U.S. Pat. Nos. 6,413,587 B1; 6,284,197 B1; and 6,288,292 B1 and Published U.S. Patent Application Nos. 2002/0004216 A1; 2002/0028451 A1; 2002/0055093 A1; 2002/0071943 A1; 2002/0142453 A1; 2002/0164604 A1; 2003/0071949 A1; and 2003/0099993 A1 for various applications employing or related to liquid crystals each of which is herein incorporated by reference in its entirety and for all purposes. U.S. Pat. No. 6,171,802 issued to Woolverton et al., and titled “Detection and Amplification of Ligands,” is directed to systems for the detection of ligands, the systems comprising at least one receptor and am amplification mechanism coupled to the receptor. An amplified signal is produced as a results of receptor binding to the ligand. Examples of suitable amplification mechanisms include antibody-embedded liquid crystalline materials; use alpha-2-macroglobulin to encage an enzyme, whereby the enzyme is separated from its substrate by a receptor; and a receptor engineered to inhibit the active site of an enzyme online in the absence of a ligand.
Many biological tools require the use of eukaryotic and prokaryotic cells. Eukaryotic mammalian cells are probably the most widely used cells although many different types of cells have found increasingly important application. Examples include, but are not limited to, tools for basic research in cell physiology, high throughput drug screening, and development sensors using cells on a chip. Lodish, H., Berk, A., Zipursky, S. L., Matsudaira, P., Baltimore, D., Darnell, J., MOLECULAR CELL BIOLOGY 4th ed., (W. H. Freeman & Company) (1999); Kapur, R., Giuliano, K. A., Campana, M., Adams, T., Olson, K., Jung, D., Mrksich, M., Vasudevan, C., Taylor, D. L., Biomed. Microdevices, 1, 99 (1999); Straub, B., Meyer, E., Fromherz, P., Nat. Biotechnol., 19, 121 (2001). Given the ubiquitous presence of the liquid crystalline state in biological systems and the technological utility of liquid crystals, it is surprising that few examples of the use of liquid crystal technologies involving whole mammalian cells has been reported. Gooby, J. W., Liquid Crystals, 24, 25 (1998); Fang, J., Ma, W., Selinger, J. V., and Shashidhar, R., Langmuir, 19, 2865 (2003). Perhaps one factor that has thus far served to prevent or limit the use of liquid crystals in conjunction with living cells is that thus far there have not been any reports of liquid crystal systems that exhibit low toxicity to living cells.
Until now, reports with respect to the toxicity of liquid crystals to cells have been very limited. Takatsu, H., Ohnishi, H., Kobayashi, K. Becker, W., Seki, M., Tazume, M., Saito, H., Sirmon-Hettich, B., Naemura, S., Mol. Cryst. Liq. Cryst., 364, 171 (2001). Toxicity tests performed by industry have thus far focused on the hazards to human health rather than toxicity to isolated cells. However, the toxicity of liquid crystals with respect to living cells is what is most relevant to applications of liquid crystals in life science and biotechnology. Therefore, a need exists for liquid crystal compositions that exhibit low toxicity towards living cell. A need also exists for compositions that include liquid crystals that may be used to culture cells, and devices for use in culturing cells with media that includes liquid crystals which exhibit low toxicity to cells. A need also exists for compositions that include a virus or a cell and a liquid crystalline compound and for methods for investigating viruses that use liquid crystals. A need further exists for methods of investigating the binding between a ligand and a receptor with mixtures of non-amphiphilic liquid crystals and the ligand.